Estabishiment Of Mutant Library And Biological Activityresearch Of Thiourea Classes And Phenyl Hydrazone Classes Compounds Based On Candida Albican FBAase

Fungal infection leads to health problems and even death,which attracts more and more attention recently.Candida albicans,as common pathogenic fungus and mode fungus,whose sugar metabolic process needs a kind of very important regulation of enzymes named type ? Fructose-1,6-bisphosphate aldolase.The enzyme can reversibly catalyze the cleavage reaction from fructose 1,6-bisphosphate(FBP)to dihydroxyacetone phosphate(DHAP)and glyceraldehyde 3-phosphate(G3P).Inhibiting the activity of FBA can block glucose metabolism process,which leads to the death of the species.In order to find Efficient,environmentally friendly,high selective inhibitors,we made some efforts to get a better understanding of the Class ? FBA.With the help of computing method such as Sybyl and some bio-chemistry methods such as site-directed mutation and enzyme kinetics test,we make a study of active sites,metal ion selectivity and new type of depressant of C.albicans type ? Fructose-1,6-bisphosphate aldolase.Specific work of this article reads as follows:1.Early,a 3D model of Ca-FBA-II was constructed by homology modeling.On this basis,we placed the substrate of small molecule with Ca-FBA-? protein cavity for docking,we found 12 amino acids interacting with the substrate,which may be important amino acids.Highly purified mutant proteins was obtained through site-directed mutagenesis technique accompanied by expression and purification technology.We tested the largest activity,binding constant,catalytic constant of the enzyme,finding that the binding constant of S62 and T290 increased more than four times,the catalytic constant of S62 and T290 reduced the more than 20 times.2.We know type ? Fructose-1,6-bisphosphate aldolase is a kind of enzyme depending on different metal ions.The selectivity of the enzyme on monovalent metal ions and bivalent metal ion was studied.Moreover,the influence of different metal ions on enzyme activity was tested when it existed solely or synergistically.Adding exogenous K+ ion in system can make the enzyme activity increase 10.5 times.Ca-FBA-? showed greater selectivity for divalent metal ions,manganese ion was best,cobalt ion and zinc ion were next.Then we measured IC50 value of good compounds on enzyme inhibition activity.3.An inhibitor screening had been made over three series of compounds including thiourea compounds,phenyl hydrazone compounds and phenylhydrazine ketone compounds at a final concentration of 50 ?M.We also tested bacteria activity and selectivity of type ? and type ? FBA of some compounds.It was found that B-15 especially showed high Ca-FBA-? activities(IC50=1.79 ?M),C-24 show high Ca-FBA-? activities(IC50=960 nM).